CN103328648B - There is the peptide of stable secondary structure and peptide storehouse and their manufacture method - Google Patents

There is the peptide of stable secondary structure and peptide storehouse and their manufacture method Download PDF

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CN103328648B
CN103328648B CN201180058284.4A CN201180058284A CN103328648B CN 103328648 B CN103328648 B CN 103328648B CN 201180058284 A CN201180058284 A CN 201180058284A CN 103328648 B CN103328648 B CN 103328648B
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mrna
sulfydryl
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营裕明
樋口岳
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University of Tokyo NUC
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Abstract

The object of the present invention is to provide the peptide etc. with stable secondary structure.The invention provides and a kind of be there is the peptide being obtained stable secondary structure by crosslinking structure, described peptide comprises the amino acid that at least one group of special acid represented in order to following formula (I) and side chain have sulfydryl, is formed with crosslinking structure by the thioether bond between the side chain of described special acid residue and described sulfydryl.In formula, (A) represent that the atomicity of singly-bound or main chain is the connection base of 1 ~ 10, (B) group containing at least one π key is represented, (C) represent hydrogen atom or the alkyl that can be substituted with a substituent, X represents the group that can be substituted by the substitution reaction based on sulfydryl.

Description

There is the peptide of stable secondary structure and peptide storehouse and their manufacture method
Technical field
The present invention relates to and a kind ofly secure peptide molecule, its synthesis by importing special acid and the secondary structure of inducing, build the method in the library be made up of the aggregate of this peptide molecule, the library of structure and the method etc. from library screening bioactive peptide.
Background technology
The peptide with α helix secondary structure, as intermolecular interactional molecular species in suppression living organism, receives publicity primarily of following 3.The first, more due to what be considered to by α spiral in molecular interaction in living organism, therefore can expect the research and development of its structure as the inhibitor of research starting point; The second, the possibility that the peptide owing to defining spirane structure has permeability of cell membrane is higher, therefore can carry out the peptide drug discovery (drugdiscovery) of intracellular protein as target; Although the 3rd is peptide, owing to obtaining the stability had proteolytic enzyme, therefore can expect to have in the blood longer than general peptide the transformation period.But only configure peptide based on the amino acid sequence information forming α spiral, most cases can not obtain these superiority.Reason is, short-chain peptide is different from the protein with tertiary structure, when the environmental exposure being rotated into spirane structure is in solvent, is subject to its considerable influence, thus can not maintains structure.Attempted repeatedly a kind of so method up to now, as its solution, namely, in order to the specificity hydrogen bond of auxiliary this spirane structure of formation, by covalently cross-linked between the amino acid side switch making suitable location, even if thus in aqueous, spirane structure also can be made to maintain or fixing.But the majority of this method utilizes chemical catalyst to form covalent linkage to the peptide chain incorporating artificial amino acid, and the whole engineering therefore comprising peptide symthesis all depends on chemosynthesis.Therefore, when requiring physiologically active for the expressing polypeptide securing α helix secondary structure, up to now majority finds drug candidate peptide by the following method, namely the sequence based on the α spiral position of known protein carries out designing or build the low centralized repository of diversity, evaluates the activity of each peptide.
On the other hand, dividing the period of the day from 11 p.m. to 1 a.m when obtaining the Toplink that is combined with particular target, widely using and carrying out the method for screening from random peptide library.Method the most general is the peptide methods of exhibiting utilizing phage, but becomes the peptide methods of exhibiting that use do not show via biological species such as intestinal bacteria in recent years.That is, as utilized various external (invitro) such as the ribosomal display method of translation or mRNA displaying methods to show method, within 1 in vitro short period of time, can build and screen the high storehouse of diversity, comparatively excellent from this point.Invitro shows method, refer to and the genotype (genotype) of phenotype (phenotype) with its sequence of coding is connected by non covalent bond or covalent linkage, thus phenotype is illustrated as genotype, the system that copies that utilization is in vitro rebuild can concentrate, increase the system of (screening (selection)) spike.The maximum feature of native system be not with protokaryon, eucaryon life entity for medium, therefore, it is possible to be separated high reactivity physiologically substance from the library with very much higher sample.As its typical comparative example, using intestinal bacteria as in the phage display of replicating medium, the screening in the library of 7 powers of diversity 10 can be carried out, and in invitro shows, the exploration that diversity reaches the library of 12 powers of 10 can be realized.Invitro displaying comprises ribosomal display, mRNA shows, RaPID shows (unexposed International Patent Application PCT/JP2010/68549) etc.Below, mRNA displaying is described as an example.
MRNA shows that method is by making polypeptide be combined with the mRNA as its template, thus by technology that the aminoacid sequence of polypeptide is mapped with nucleotide sequence.For this reason; 3 ' the end of mRNA will be attached to as the tetracycline of acidylate tRNA end analogue by suitable connexon; it is made to participate in translation reaction; tetracycline enters into ribosomal A position thus; covalent linkage is formed with the peptide in extending; its result, the peptide molecule of translation product is connected with mRNA (patent documentation 1 ~ 3, non-patent literature 1,2) by tetracycline.
So, in invitro shows, although the multifarious peptide storehouse of 12 powers had up to 10 can be screened, build peptide storehouse owing to utilizing the function of organism, therefore, the peptide storehouse be only made up of Argine Monohydrochloride can only be constructed.In order to overcome the low problem of this integrant, as long as can by the New function do not had in natural function being incorporated into the peptide storehouse building in amino acid structure and there is the Novel framework that normally unstable α helix secondary structure can be made stable, screen, then can expect, the peptide showing highly inhibited, the function such as selectivity, stability do not realized as naturally occurring simple peptide chain can be obtained.
In recent years, along with the development of technology being called " expansion of genetic code " or " reprogrammed of genetic code ", by comprising the various displaying methods of phage display, to manufacture and screening has special amino acid whose peptide storehouse, in fact becoming possibility.
In the expansion of genetic code, utilize the terminator codon or artificial four base codon that are not used to designated amino acid in natural translation system, distribute special acid to these codons, thus achieve and comprise the protein of special acid, the synthesis of peptide.But the limited amount of terminator codon or available four base codon, causes the quantity of available special acid simultaneously to there is the upper limit (in fact less than 3 kinds).
About importing the research in order to the peptide of the crosslinking structure for the purpose of stable alpha spiral, also actively develop.At present, main report has following method: the method based on amido linkage, the method based on disulfide bond and the method (non-patent literature 3) formed based on the alkene by Ring closing metathesis.By importing these covalent linkage and induced synthesis spirane structure, but can usually there is organism internal stability problem.That is, for amide structure or curing structure, easily rupture because of proteolytic enzyme or reductive condition, be therefore unfavorable for utilizing as inhibitor.What overcome this point is the method formed based on alkene, and the known peptide having imported this crosslinking structure has activity (non-patent literature 4) in vivo.At present, the research and development of the physiologically active peptide that make use of the crosslinking structure formed based on this alkene are carried out.
But build with after hangar from known aminoacid sequence by chemosynthesis, need to carefully study the crosslinking structure position not losing physiologically active, therefore its research and development need to drop into very large energy.And based in the exploring with hangar of such as above-mentioned design, the peptide sequence do not obtained may be the confirmation of optimal sequence.It's a pity, due to the above-mentioned crosslinking reaction formed based on alkene, use chemical catalyst, be therefore difficult to be suitable for invitro and show method.That is, due under the state that must coexist at the necessary rrna of translation reaction or the factor such as protein, ATP, carry out alkene formation, therefore there is the hidden danger that its reaction efficiency is low, and it is comparatively difficult to build high-quality library.Therefore, in order to the target to more wide region, find the physiologically active expressing polypeptide making αhelix fixing, must carry out being suitable for the research and development that invitro shows the cross-linking method of method.This cross-linking method needs rapid under translation system condition and highly selective carries out crosslinking reaction, and its crosslinking structure formed must be the structure being not easy in organism to be decomposed.
Prior art document
Patent documentation
Patent documentation 1: Japanese Patent No. 3683282 publication (International Publication WO98/16636)
Patent documentation 2: Japanese Patent No. 3683902 publication (International Publication WO98/31700)
Patent documentation 3: Japanese Patent No. 3692542 publication
Non-patent literature
Non-patent literature 1:Roberts etc., Proc.Natl.Acd.Sci.USA, 1997,94,12297-12302
Non-patent literature 2:Nemoto etc., FEBSLett., 1997,414,405-408
Non-patent literature 3:Taylor, W., J., Baum, J. etc., J.Am.Chem.Soc., Vol.116, p.6431-6432 (1994), Schultz, P.G. etc., J.Am.Chem.Soc., Vol.113, p.9391-9392 (1991), Phelan, J.C. etc., J.Am.Chem.Soc., Vol.119, p.455-460 (1197), Grubbs, R.H., Blackwell, H.E., Angew.Chem.Int.Ed.Vol.37, p.3281-3284 (1998)
Non-patent literature 4:Verdine, G.L., Korsmeyer, S.J. etc., Science, Vol305, p.1466-1470 (2004)
Summary of the invention
Technical problem
The object of the invention is to, providing by importing to peptide chain translation the peptide etc. that the special acid being designed to spontaneous formation crosslinking structure obtained, had stable secondary structure under translation synthesis condition.
Technical scheme
Utilize the expressing polypeptide synthetic technology based on genetic code reprogrammed developed in recent years by inventors of the present invention, synthesis has the expressing polypeptide of the side chain that can react containing the halfcystine of sulfydryl (sulfanylgroup) (-SH base) or its analogue with side chain, and the expressing polypeptide (Fig. 1) securing object secondary structure is synthesized in translation.
Before describing the present invention in detail, first " reprogrammed of genetic code " that become background technology is simply introduced below.
In the translation of living organism, the arrangement (triplet (triplet)) of three bases of mRNA determines an amino acid as a codon, the peptide of synthesis its arrangement corresponding.Now, by following two stages, codon is mapped with amino acid.I corresponding amino acid is incorporated into tRNA end by () aminoacyl tRNA synthetase (aminoacyl-tRNAsynthetase:ARS).(ii) anticodon of tRNA matches with the codon of corresponding mRNA, thus along the information of mRNA and amino acid on tRNA be polymerized, synthetic peptide.
Corresponding relation between this codon and anticodon, almost general, 64 kinds of codons are assigned any one of 20 seed amino acids respectively.Standard genetic code table is shown below.
[table 1]
But, utilize rebuilding type translation system and artificial aminoacylates RNA enzyme in conjunction with Proteinase, bone marrow serine (flexizyme), can this genetic code of reprogrammed.
Rebuilding type translation system (reconstitutedtranslationsystem), refer to that separation and purification respectively such as rrna, translation factor, tRNA, amino acid and ATP or GTP equal energy source etc. synthesize the relevant factor with the translation of protein or peptide, carry out the translation system mixed.Such as, as utilizing colibacillary ribosomal system, the technology be recorded in document is below known: H.F.Kung, B.Redfield, B.V.Treadwell, B.Eskin, C.Spears and H.Weissbach (1977) " DNA-directedinvitrosynthesisofbeta-galactosidase.Studies withpurifiedfactors " TheJournalofBiologicalChemistryVol.252, No.19,68896894; M.C.Gonza, C.Cunningham and R.M.Green (1985) " IsolationandpointofactionofafactorfromEscherichiacolireq uiredtoreconstructtranslation " ProceedingofNationalAcademyofSciencesoftheUnitedStatesof AmericaVol.82,1648-1652; M.Y.Pavlov and M.Ehrenberg (1996) " RateoftranslationofnaturalmRNAsinanoptimizedinVitrosyste m " ArchivesofBiochemistryandBiophysicsVol.328, No.1,9-16; Y.Shimizu, A.Inoue, Y.Tomari, T.Suzuki, T.Yokogawa, K.Nishikawa and T.Ueda (2001) " Cell-freetranslationreconstitutedwithpurifiedcomponents " NatureBiotechnologyVol.19, No.8,751-755; H.Ohashi, Y.Shimizu, B.W.Ying and T.Ueda (2007) " Efficientproteinselectionbasedonribosomedisplaysystemwit hpurifiedcomponents " BiochemicalandBiophysicalResearchCommunicationsVol.352, No.1,270-276.
On the other hand, in conjunction with Proteinase, bone marrow serine (flexizyme), it is the engineered rna enzyme (there is the RNA enzyme of acidylate tRNA synthetic enzyme sample activity) that can connect (acidylate) arbitrary amino acid or alcohol acid on arbitrary tRNA.Such as, the enzyme be recorded in following document is known: H.Murakami, H.Saito and H.Suga, (2003), Chemistry & Biology, Vol.10,655-662; H.Murakami, D.Kourouklis and H.Suga, (2003), Chemistry & Biology, Vol.10,1077-1084; H.Murakami, A.Ohta, H.Ashigai, H.Suga (2006) NatureMethods3,357-359 " Theflexizymesystem:ahighlyflexibletRNAaminoacylationtool forthesynthesisofnonnaturalpeptides "; N.Niwa, Y.Yamagishi, H.Murakami, H.Suga (2009) Bioorganic & MedicinalChemistryLetters19,3892-3894 " Aflexizymethatselectivelychargesaminoacidsactivatedbyawa ter-friendlyleavinggroup "; And WO2007/066627 " multi-functional acylase and uses thereof ".Also known by people in conjunction with titles such as Proteinase, bone marrow serines (aminoflexizyme) in conjunction with Proteinase, bone marrow serine (enhancedFlexizyme:eFx), amino in conjunction with Proteinase, bone marrow serine (dinitrobenzylFlexizyme:dFx), enhancement type in conjunction with Proteinase, bone marrow serine (Fx) and from the dinitrobenzene benzyl that prototype changes in conjunction with Proteinase, bone marrow serine with prototype in conjunction with Proteinase, bone marrow serine.
Or, the additive method such as aminoacylates based on chemical process can also be used, as a kind of deformation method that can connect arbitrary amino acid whose method on arbitrary tRNA.
In the reprogrammed of genetic code, utilize and freely accept or reject the Components of translation system and only the recombinate composition and the translation system set up out that need according to object.Such as, specific amino acid whose translation system is eliminated, then to becoming blank codon by amino acid whose codon if rebuild.Then; utilization (or utilizes the aminoacylates (chemicalaminoacylation) of chemical reaction in conjunction with Proteinase, bone marrow serine; or utilize the aminoacylates of mutant proteases); special acid is connected to the tRNA of the anticodon with blank codon complementation with this, adds that this amino acid is translated.Accordingly, special acid is encoded by this codon, thus produces the amino acid of replacement removal by translating and imported the peptide of special acid.
As a kind of non-limiting implementation of the present invention, by the reprogrammed technology of genetic code, such as, synthesize the expressing polypeptide that side chain has propargyl chloride (propargylchloride) structure.Now, if clip the residue of suitable number from the position of the residue with above-mentioned side-chain structure and configure cysteine residues in peptide, then spontaneously on propargyl position because of its sulfydryl carry out substitution reaction after translation, between peptide side chain, form crosslinking structure by thioether (thioether) key.That is, by integrating the structure that a group can form key in amino acid, namely as propargyl chloride structure and the sulfydryl as nucleophilic reagent of electrophilic reagent, can key be formed in-between and build crosslinking structure.This crosslinking structure is assisted the location specific hydrogen bond of the key becoming spiralization and induces spirane structure, gives the secondary structure that peptide is expected.This molecular structure group forming key is not limited to propargyl chloride structure and halfcystine.Describe in detail below.
Further, the invention provides one and utilize above-mentioned technology and build peptide storehouse, obtain the technology of the inhibitor of molecular interaction in living organism from this peptide storehouse.
Main points of the present invention are as follows.
(1) peptide, this peptide has and obtains stable secondary structure by crosslinking structure,
Described peptide comprises the amino acid that at least one group of special acid represented by following formula (I) and side chain have sulfydryl,
[chemical formula 1]
(in formula, (A) represent that the atomicity of singly-bound or main chain is the connection base of 1 ~ 10, (B) group containing at least one π key is represented, (C) represent hydrogen atom or the alkyl that can be substituted with a substituent, X represents the group that can be substituted by the substitution reaction based on sulfydryl.〕
Described peptide is formed with crosslinking structure by the thioether bond between the side chain of described special acid residue and described sulfydryl.
(2) peptide as described in above-mentioned (1), in described formula (I),
(A) from by singly-bound; Can be substituted with a substituent, carbonatoms be 1 ~ 10 alkylidene group, carbonatoms be 2 ~ 10 alkenylene or carbonatoms be the alkynylene of 2 ~ 10; 1 ~ 2 carbon atom of main chain is replaced by Sauerstoffatom, nitrogen-atoms or sulphur atom, can be substituted with a substituent, the atomicity of main chain is select in the group that forms of alkylidene group, alkenylene or alkynylene of less than 10,
(B) from by-C ≡ C-,-C=C-,-Ar-,-Ar-Ar-,-Ar-C ≡ C-,-Ar-C=C-,-NHC (O)-,-C (O)-,-Ar-NHC (O)-, select the group of-Ar-C (O)-form,
(C) select from the group be made up of hydrogen atom, methyl, ethyl, propyl group, butyl and amyl group,
X is from by Cl, Br, I ,-OSO 2me, tosyl group (tosyl), nitrobenzenesulfonyl (nitrobenzenesulfonyl:Nosyl) ,-OSO 2(R represents from by CH-Ar-R 3, NO 2, CF 3and the group selected in the group of H formation.) select in the group that forms.
(3) peptide as described in above-mentioned (1) or (2), the special acid of described formula (I) represents by with the individual arbitrarily of following formula (II) ~ (IV),
[chemical formula 2]
(in formula, m represents the integer selected from 1 ~ 10, and X is from by Cl, Br, I ,-OSO 2me, tosyl group, nitrobenzenesulfonyl ,-OSO 2(R represents from by CH-Ar-R 3, NO 2, CF 3and the group selected in the group of H formation.) select in the group that forms.〕
(4) peptide as described in any one in above-mentioned (1) to (3), the described amino acid with sulfydryl is selected from by halfcystine and the group that forms in order to the cysteine analogs that lower formula V and (VI) represent,
[chemical formula 3]
(in formula, m represents the integer selected from 1 ~ 10.〕
(5) peptide as described in any one in above-mentioned (1) to (4), described special acid residue and described side chain have the amino-acid residue of sulfydryl, in each group, interval 2,3,6 or ten amino acid residue and configure.
(6) method, preparation has the peptide being obtained stable secondary structure by crosslinking structure, comprises following operation:
(i) be configured with in synthetic molecules by translation at least one group of side chain have sulfydryl amino acid and by with following formula (I)
[chemical formula 4]
(in formula, (A) represent that the atomicity of singly-bound or main chain is the connection base of 1 ~ 10, (B) group containing at least one π key is represented, (C) represent hydrogen atom or the alkyl that can be substituted with a substituent, X represents the group that can be substituted by the substitution reaction based on sulfydryl.) expressing polypeptide of special acid that represents; And
(ii) in each described group, the side chain of the special acid of described sulfydryl and formula (I) is formed crosslinking structure by thioether bonding.
(7) method as described in above-mentioned (6), described side chain has the amino acid of sulfydryl and the special acid of formula (I), in each group, interval 2,3,6 or ten amino acid residue and be configured.
(8) method as described in above-mentioned (6) or (7), the translation synthesis procedure of described (i) comprising: utilize and the special acid of formula (I) is connected to tRNA and the aminoacyl tRNA that obtains, and translation has the template mRNA of the change codon of this special acid of coding.
(9) method as described in above-mentioned (8), the amino acid that described side chain has sulfydryl is by the special acid changing codon coding, the translation synthesis procedure of described (i) comprising: utilize this special acid special acid of formula (I) and side chain with sulfydryl to be connected to tRNA and the aminoacyl tRNA that obtains, and translation has the template mRNA of the change codon of special acid of encoding respectively.
(10) method as described in above-mentioned (8) or (9), described aminoacyl tRNA is by utilizing the RNA enzyme with acidylate tRNA synthetic enzyme sample activity, special acid being connected to tRNA and obtaining.
(11) method as described in any one in above-mentioned (6) to (10), the special acid represented by described formula (I), represents by with the individual arbitrarily of following formula (II) ~ (IV),
[chemical formula 5]
(in formula, m represents the integer selected from 1 ~ 10, and X is from by Cl, Br, I ,-OSO 2me, tosyl group, nitrobenzenesulfonyl ,-OSO 2(R represents from by CH-Ar-R 3, NO 2, CF 3and the group selected in the group of H formation.) select in the group that forms.〕
(12) method as described in any one in above-mentioned (6) to (11), the described amino acid with sulfydryl is selected from by halfcystine and the group that forms in order to the cysteine analogs that lower formula V and (VI) represent,
[chemical formula 6]
(in formula, m represents the integer selected from 1 ~ 10.〕
(13) a peptide storehouse, comprises two or more peptide as described in any one in above-mentioned (1) to (5).
(14) the peptide storehouse as described in above-mentioned (13), each described peptide is connected with its mRNA of coding.
(15) method, builds the peptide storehouse as described in above-mentioned (13), comprises following operation:
I () prepares mRNA library, this mRNA library comprises the codon of special acid that at least one group of amino acid whose codon and determining determining that side chain has sulfydryl is represented by the formula (I) described in above-mentioned (1) in the RNA of coding random amino acid sequence, interval 2,3,6 or ten amino acid unit in each group and be configured with and determine that side chain has the codon of the amino acid whose codon of sulfydryl and the special acid of decision formula (I);
(ii) by the cell free translation system containing the tRNA be connected with described special acid, translate described mRNA, obtain the aggregate of the peptide being configured with described special acid in stochastic sequence; And
(iii) in each peptide, make the side chain bonding of the special acid of sulfydryl and formula (I) and form crosslinking structure.
(16) method, builds the peptide storehouse as described in above-mentioned (14), comprises following operation:
In the operation (i) as described in above-mentioned (15), further, to the 3' end of mRNA in conjunction with tetracycline, obtain tetracycline in conjunction with mRNA library;
In the operation (ii) as described in above-mentioned (15), described tetracycline is expressed in conjunction with mRNA library in cell free translation system, obtain the peptide-mRNA mixture being configured with described special acid in stochastic sequence; And
Carry out the operation (iii) as described in above-mentioned (15).
(17) method as described in above-mentioned (15) or (16), the change codon of the special acid of decision formula (I) is AUG codon, mRNA stochastic sequence by any triplet in NNC or NNU sequence (N represents any one base in A, U, G, C) repeat form.
(18) a kind of special acid represented by following formula (I),
[chemical formula 7]
(in formula, (A) represent that the atomicity of singly-bound or main chain is the connection base of 1 ~ 10, (B) group containing the double bond between at least one carbon atom, the group containing the triple bond between at least one carbon atom or the group containing at least one aromatic nucleus is represented, (C) represent hydrogen atom or can have the substituent alkyl that carbonatoms is 1 ~ 3, X represents the group that can be substituted by the substitution reaction based on sulfydryl.〕
(19) method, select the peptide be combined with target proteins from the peptide storehouse as described in above-mentioned (13) or (14), comprise following operation:
I peptide storehouse contacts with target proteins by (), carry out incubation; And
(ii) peptide molecule be combined with target proteins is selected.
(20) method, select the peptide molecular interaction of target proteins to inhibit activities from the peptide storehouse as described in above-mentioned (13) or (14), comprising:
(first) primary screening, comprises the operation (i) as described in above-mentioned (19) and (ii), selects the peptide be combined with target proteins; And
(second) postsearch screening, for the inhibit activities of the molecular interaction to target proteins of the peptide selected by primary screening, is evaluated, and determines that described peptide is the peptide molecular interaction of target proteins to inhibit activities.
(21) method, manufactures the peptide be combined with target proteins, comprises following operation:
I peptide storehouse as described in above-mentioned (14) contacts with target proteins by (), carry out incubation;
(ii) peptide-mRNA mixture be combined with target proteins is selected;
(iii) mRNA of the peptide-mRNA mixture selected by amplification, obtains peptide-mRNA mixture;
(iv) operation (i) ~ (iii) is repeated once, the peptide-mRNA mixture that concentrated affinity is high; And
(v) from the mRNA by the concentrated peptide-mRNA mixture of operation (iv), expression of peptides.
(22) method as described in any one in above-mentioned (19) to (21), target proteins is the molecule of inhibited apoptosis.
Beneficial effect
Can be obtained by the present invention and on the position expected, import crosslinking structure and there is the peptide of stable secondary structure (such as αhelix).Because this peptide has stable secondary structure, therefore in the performance that common peptide can not have, such as superior to the high-affinity of target, highly selective, permeability of cell membrane, stability aspect.
Further, prepare the randomized peptide storehouse of aminoacid sequence in this peptide, use this peptide storehouse to screen, can obtain thus and to target proteins etc., there is the peptide of high-affinity or suppress the peptide etc. of function of target proteins to have the peptide of physiologically active.
Further, in peptide storehouse, be connected by peptide and its nucleic acid of coding and be suitable for invitro and show method, concentrating and increase and becoming easy of selected peptide can be made thus.
Accompanying drawing explanation
Fig. 1 illustrates the one example of the expressing polypeptide securing α helix secondary structure.
Fig. 2 illustrates the configuration of crosslinking structure precursor.
Fig. 3 illustrates the formation of selective crosslinking structure.
Fig. 4 summarizes the screening illustrating and make use of the crosslinked peptide storehouse shown according to mRNA.
Fig. 5 illustrates the structure example in mRNA library.
Fig. 6 summarizes the translation illustrated based on changing genetic codon table.
Embodiment
import the peptide of crosslinking structure
The invention provides and a kind of there is the new peptides obtaining stable secondary structure because possessing crosslinking structure.In this manual, " secondary structure ", is referred to and to be formed by the hydrogen bond in peptide main chain, compare the special construction observed in narrow range, refer to spiral or beta structure.The present invention is applicable to α spiral, 3 10the stabilization of the spirane structures such as spirane structure, but other secondary structures also can be made to stablize.
Further, in the description, make " secondary structure is stablized " of peptide, refer to that the degree that secondary structure can be combined with target molecules with peptide is stablized, this combination has circulation ratio to a certain degree.
If make secondary structure stablize by importing crosslinking structure to peptide, then think that (i) can improve protease resistant, (ii) can improve membrane permeability, (iii) can improve the affinity with target proteins.As described in the background art, up to now, make between amino acid side chain crosslinked by amido linkage or disulfide linkage, attempt the secondary structure of stabilized peptide.Further, also report has the crosslinking reaction based on utilizing the alkene of chemical catalyst to be formed.
But different from method up to now, the crosslinking structure formed in peptide of the present invention is not easy point formal similarity in body, specifically, can not decompose as amido linkage because of enzyme, also can not be reduced as disulfide linkage.
Peptide of the present invention, comprise the amino acid that at least one group of special acid represented by following formula (I) and side chain have sulfydryl (sulfanylgroup), be formed with crosslinking structure by the thioether bond between the side chain of special acid and sulfydryl.
[chemical formula 8]
In formula, (A) represents the position played a role as interval (spacer).(A) be the connection base that the atomicity of singly-bound or main chain is 1-10, as long as (B) described later CH 2crosslinking reaction between-X and sulfydryl is suitable carries out, whatsoever group, but such as can enumerate, singly-bound; That can be substituted with a substituent, that carbonatoms is the alkylidene group (alkylenegroup) of 1-10, carbonatoms is 2-10 alkenylene (alkenylenegroup) or carbonatoms are the alkynylene (alkynylenegroup) of 2-10; 1-2 carbon atom of main chain is replaced by Sauerstoffatom, nitrogen-atoms or sulphur atom, can be substituted with a substituent, the atomicity of main chain be less than 10 alkylidene group, alkenylene or alkynylene; Deng, but be not limited thereto.
Wherein, when synthesizing peptide of the present invention by translation synthesis method, what also have (A) has the advantage being beneficial to translation reaction and carrying out.
For (B), as long as induction is based on the substitution reaction of the X of sulfydryl, whatsoever group, but can be such as the group comprising at least one π key.As the group containing π key, can be following group: the alkylidene group that can be substituted with a substituent, alkenylene or alkynylene; The group containing at least one aromatic nucleus that can be substituted with a substituent; Group containing at least one ketone group or amide group.For the carbonatoms of (B), when alkylidene group, alkenylene, alkynylene, such as, can be 1-10,2-8 is individual, 2-5 is individual.When the group containing at least one aromatic nucleus, group containing at least one ketone group or amide group, the atomicity of main chain can be such as 1-10,2-8,2-5 etc.For the position of π key, as long as make the substitution reaction based on the X of sulfydryl be suitable for carrying out, be not particularly limited, but preferably, at least one atom forming π key with can be combined by the carbon atom carried out based on the substitution reaction of sulfydryl, the most preferably, clip CH from X 2group and directly have π key.
As the example of (B), can enumerate-C ≡ C-,-C=C-,-Ar-,-Ar-Ar-,-Ar-C ≡ C-,-Ar-C=C-,-NHC (O)-,-C (O)-,-Ar-NHC (O)-,-Ar-C (O)-, but to be not limited thereto.
Below, illustrate by the thioether bond between sulfydryl and the side chain of special acid represented by formula (I) and the example of the crosslinking structure formed.
[table 2]
For X, such as, as long as by departing atom or group based on the substitution reaction of sulfydryl, being not particularly limited, can be Cl, Br, I ,-OSO 2me ,-OSO 2(in formula, R represents from by CH-Ar-R 3, NO 2, CF 3and the group selected in the group of H formation).
(C) hydrogen atom or the alkyl that can be substituted with a substituent is represented.Such as, can be methyl, ethyl, propyl group, butyl, the amyl group that can be substituted with a substituent, also can be side chain.
In this manual, alkenylene refers in main chain the alkylene with 1-3 double bond, and alkynylene refers in main chain the alkylene with 1-3 triple bond.For the position of double bond or triple bond, as long as peptide reaches desired effect, be not particularly limited.
In this manual; for substituting group; although be not particularly limited; but can enumerate, alkoxyl group, cyano group, nitro, hydroxyl, carboxyl, acyl group, amino, aryl, heteroaryl, phenoxy group, non-aromatic heterocycle etc. that the alkyl that halogen atom, the carbonatoms that can be substituted are 1-6, the carbonatoms that can be substituted are 1-6.
For peptide of the present invention, owing to forming key between the pendant moiety of the special acid of formula (I) and sulfydryl and forming crosslinking structure, therefore amino acid-the residue of the key element of this peptide is formed as a unit, these group of functional group needs to be configured to be present in the unit of different integrants, when forming crosslinking structure, secondary structure is stablized.For convenience of explanation, the unit of this integrant is called amino acid unit.
The amino acid unit (being equivalent to special acid) becoming electrophilic reagent and the amino acid unit with sulfydryl are preferably configured in use (i, i+3), (i, i+4), (i, i+7) or (i, i+11) position of representing.Please refer to Fig. 2.At this, i is the arbitrary integer of more than 0, is the position of the amino acid unit counted from the residue of the N-terminal of the peptide comprising cross-linking precursor.Such as, during i=3, (i, i+4) represents, be configured as the amino acid unit of electrophilic reagent in the position of 3 amino acid unit calculated from N-terminal (the 0th) and the position of 7 amino acid unit and there is the amino acid unit of sulfydryl, forming crosslinking structure.
As i+3 or i+4, if α spiral then occurs crosslinked on 1 circle, as i+7 or i+11, occur crosslinked respectively on 2 circles, 3 circles.In other words, such as, preferably 2,3,6 or ten amino acid unit be present in formed crosslinking structure amino acid unit between.
Between the amino acid unit forming crosslinking structure and the amino acid unit of front and back, except the initial amino acid of N-terminal, be preferably in molecule and there is amino (-NR 2) and the arbitrary amino acid of compound (aminocarboxylic acid (aminocarboxylicacid)) of this Liang Ge functional group of carboxyl (-COOH), alpha-amino carboxylic acid typically, more preferably Argine Monohydrochloride.For become electrophilic reagent amino acid unit and have sulfydryl amino acid unit configuration for, whichever is configured in N-terminal side or C-terminal side.
Special acid, refer to and 20 kinds of Argine Monohydrochloride ratios using in natural translation, no matter all amino acid that structure is different is synthetic, or nature exists.That is, all can comprise: the non-protein amino acid that a part for the side-chain structure of Argine Monohydrochloride is changed by chemical reaction or modifies or artificial amino acid, D type amino acid, N-methylamino acid (N-MethylAminoAcid), N-acylamino acid (N-acylaminoacid), beta-amino acids, there is the derivative etc. of the structure that amino on amino acid backbone or carboxyl are substituted.Expressing polypeptide, refer to containing more than one special acid, two or more amino acid is mainly with the peptide that peptide bond (also comprise and there is ring texture or ester bond) combines.
In the present invention, as the amino acid whose typical example with sulfydryl, halfcystine or its analogue (cysteine analogs (cysteineanalog)), as can with the amino acid whose typical example becoming electrophilic reagent of its reaction, be the special acid of formula (I).As hereinafter described, for the amino acid with sulfydryl, when it is halfcystine, can standard codon be used, but when being non-protein amino acid, be encoded by changing codon.On the other hand, the special acid of formula (I) is generally encoded by changing codon.
Further, in the present invention, the initial amino acid becoming peptide chain N-terminal is also not limited to methionine(Met), can be the arbitrary Argine Monohydrochloride beyond methionine(Met) or arbitrary special acid.Such as, in embodiment described later, build the translation system removing methionine(Met) from common 20 seed amino acids, replace and use the tRNA of connection N α-acetylated amino acids on initial tRNA and start translation.This N-terminal is modified and can be said for guaranteeing that the stability of peptide is effective.Or two amino acid whose one that forms cross-linking precursor also can be initial amino acid (that is, when i is 0).
Form the amino acid whose more typical mode of cross-linking precursor about configuration, be all imported by extension by these amino acid, be configured to the mode of peptide chain inside, that is, i is the situation of the integer of more than 1.Further, the size being applicable to peptide of the present invention is not particularly limited, but the present invention is particularly useful for generally being difficult to the peptide that maintains below 25 amino-acid residues of spirane structure.
As the concrete example of the compound of formula (I), such as, the compound of formula (VII) can be enumerated:
[chemical formula 9]
In formula, m represents the integer of 1 to 10.As the concrete example of the compound of formula (VII), the compound that m is 1 can be enumerated, this compound such as can utilize Schollkopf chiral auxiliary(reagent) (chiralauxiliary) and with the excessive manufacture of high antimer.
Further, the compound of formula (VIII) can also be enumerated:
[chemical formula 10]
In formula, the definition of m is identical with above-mentioned.
Further, the compound of formula (IX) can also be enumerated:
[chemical formula 11]
In formula, the definition of m is identical with above-mentioned.As the concrete example of the compound of formula (IX), the compound that m is the following formula (X) of 1 can be enumerated.
[chemical formula 12]
Further, as the concrete example of the compound that (C) in formula (I) is alkyl chain, the formula (XI) of corresponding (VII) can be enumerated, the compound of the formula (XII) of corresponding (X):
[chemical formula 13]
On the other hand, as the concrete example of cysteine analogs, such as, can enumerate the compound of formula (V) or formula (VI).
[chemical formula 14]
In formula (V), (VI), the definition of m is identical with above-mentioned.
Further, the special acid that homocysteine or sulfydryl norvaline (mercaptonorvaline) etc. have sulfydryl can also be enumerated.
import the manufacture method of the peptide of crosslinking structure
Then, the manufacture method of peptide of the present invention is described.
Peptide of the present invention can utilize based on chemosynthesis method or use the various known method such as method of translation synthesis system or based on these methods method and manufacture.Such as; when using chemical synthesis process; can by liquid phase synthesis, employ the solid phase synthesis of the blocking groups such as Fmoc or Boc, be combined with the various method such as hybrid system of liquid phase synthesis and solid phase method, synthesis is containing the special acid of formula (I) and the amino acid whose peptide with sulfydryl.If synthesized peptide, then as hereinafter described, without the need to using enzyme etc., between the side chain of the special acid of formula (I) and sulfydryl, spontaneous formation thioether bond, can obtain crosslinked peptide.
As the one example of the manufacture method of peptide of the present invention, below illustrate and utilize cell free translation synthesis system and the method that manufactures.The method utilizes the highly selective intramolecular reaction participated in by the special acid comprised in the peptide of translation synthesis, between peptide side chain, form covalent linkage, builds crosslinking structure, comprises following operation.
(i) synthesize by translating at least be configured with in a molecule side chain have the group of the amino acid of sulfydryl and the special acid represented by formula (I), expressing polypeptide; And
(ii), in described each group, the side chain of the special acid of described sulfydryl and formula (I) is formed crosslinking structure by thioether bonding.
Operation (i) is called translation synthesis procedure, operation (ii) is called crosslinking structure formation process.
In the translation synthesis procedure of operation (i), the peptide that two seed amino acids below being obtained by translation synthesis are only separated suitable total number of atnino acid and configure:
(first) side chain has the amino acid of sulfydryl, and
Special acid represented by (second) formula (I).
Below, in expressing polypeptide synthesized in operation (i), by containing by sulfydryl and the chemical structure of one group of functional group that can form with the functional group of the special acid of its reaction, be called cross-linking precursor.That is, only separate that suitable total number of atnino acid configures, these (first) and (second) amino acid whose group, form cross-linking precursor.
Such as, please refer to the mode chart of Fig. 2.In fig. 2, these amino acid whose group, represent with the one group of aterrimus circle being configured in peptide chain inside.Further, the cross-linking precursor that a peptide chain can comprise, not only can have one group, can also configure multiple cross-linking precursor.If the peptide chain containing multiple cross-linking precursor has been synthesized in translation, then, in the crosslinking structure formation process of operation (ii), multiple crosslinking structure can be formed.
The special acid of formula (I) has and the structure of key forming reactions can occur, as the side-chain structure that can be formed for crosslinked covalent linkage with the sulfydryl (-SH) of nucleophilic reagent functional group.As the result of this reaction, in the crosslinking structure formation process of operation (ii), the peptide comprising cross-linking precursor becomes the peptide being formed with crosslinking structure.In the present invention, form crosslinking structure from cross-linking precursor, be automatically occur under translation synthesis condition, therefore do not need chemical catalyst.
When carrying out operation (i) under cell free translation system, utilizing genetic code reprogrammed technology, importing in peptide chain by distributing special acid artificially in known codon.Specifically, preparation has the mRNA of the codon of coding special acid, by this mRNA is translated under change genetic codon table, can obtain and be imported into the peptide of special acid on the position of being specified by reformed codon (change codon).
In this application, codon refers to these the two kinds of codons of standard codon changing codon and use in natural translation.Change codon, refer to the corresponding relation being eliminated Argine Monohydrochloride by genetic code reprogrammed, the codon be mapped with special acid.Special acid can only by changing codon coding.
By importing crosslinking structure in the peptide compounds of synthesis as mentioned above, synthesis helical peptides compound.For the formation of the reaction conditions of crosslinking structure, can set according to the kind of the group of functional group, but generally for the synthesis of under the cell free translation system condition of this peptide compounds, highly selective reacts.Therefore, do not need to adjust reaction conditions especially, can spontaneous formation key and obtain crosslinked peptide compounds.
Such as, please refer to Fig. 3.Translation is started by formylmethionine (formylmethionine), the various amino acid whose combinatorial compound be imported into by extension codon is with (i, the peptide chain of position relationship configuration i+4), its result, only by the special acid X of formula (I) and the group of halfcystine C, by thioether bond, form crosslinking structure.
Similarly, in embodiment described later, record and synthesize by translation the peptide that the peptide sequence being configured with the special acid and halfcystine comprising the propargyl chloride being configured in amino acid side chain obtained, comprised cross-linking precursor.In exemplary mode, after translation, the spontaneous substitution reaction based on sulfydryl carried out to propargyl chloride position of meeting, forms thioether bond and completes crosslinking structure.
acellular (invitro) translation system
Translation system is the place for translating synthetic peptide, is to comprise both concept of usual method and test kit (thing).Cell free translation system used in the present invention, preferably utilizes the known rebuilding type translation system of segmentation further, builds the system that impurity is the least possible.More existing system, is described the concrete constituent of the translation system as the available test kit of the present invention (thing).
As the concrete example of the constituent of translation system, comprise rrna, IF group, EF group, RF group, RRF, in order to synthesize object peptide and supporting group of required MIN natural amino acid, tRNA, specificity ARS proteolytic enzyme, the energy etc. for translation reaction.
As rrna, can be suitable for utilizing the rrna from intestinal bacteria separation and purification.
Protein-based use translation initiation factor (such as, IF1, IF2, IF3), translation elongation factor (such as, EF-Tu, EF-Ts, EF-G), the translation termination factor (such as, RF1, RF2, RF3, RRF), for enzyme (such as, creatine kinase (creatinekinase), myokinase (myokinase), Pyrophosphate phosphohydrolase (pyrophosphatase), the NDPK (nucleotide-diphosphatasekinase) of energy regeneration.Wherein, the translation termination factor, enzyme for energy regeneration, can add as required.In order to carry out transcribing from template DNA, sometimes also add t7 rna polymerase, but when adding to translation system the mRNA transcribed in advance, without the need to adding RNA polymerase.
In addition, can be suitable for using in the same manner as existing system: suitable buffered soln, the NTP as the energy of translation reaction, phosphocreatine (creatinephosphate), for activating rrna, stablize RNA, the necessary factor etc. of stabilizing protein.And, in common translation reaction, because initiator codon AUG determines N-formylmethionine (N-formyl-methionine) by initial tRNA, therefore must just like 10-formyl-5,6,7,8-tetrahydrofolic acid (THFA) (10-formyl-5,6,7,8-tetrahydroforlicacid) (Baggott etc., 1995) the formyl donor (formyldonor) such as, but in the present invention, when starting translation reaction by special acid, formyl donor is not required.According to same reason, Methionyl-tRNA formyltransferase (methionyl-tRNAformyltransferase, MTF), neither be required.
In the translation system that the present invention utilizes, for natural Argine Monohydrochloride, corresponding natural tRNA and ARS can be utilized as existing system.As the example of natural tRNA, enumerate and gather intestinal bacteria and carry out fragmentation and the mixture of intestinal bacteria purifying tRNA molecule (tRNAfraction) from fragmentation, also can buy commercially produced product.Specific A, U, C, G in natural tRNA are modified by sulphation by enzyme.Or also can use the tRNA with the native sequences of in vitro transcribing.
On the other hand, for special acid, preferably to utilize be not natural tRNA, artificial tRNA as the tRNA transcription product of orthogonal tRNA (orthogonaltRNA).Artificial tRNA can pass through with DNA to be that template utilizes external (invitro) responsive transcription of suitable RNA polymerase to prepare.Chemically modified is there is not completely in this artificial tRNA.
In order to the peptide to translation product imports special acid, add in advance by the orthogonal tRNA of special acid acidylate to translation system.In preferred mode; prepared by the following method by the tRNA of special acid acidylate: under the non-existent condition of other tRNA or ARS; special acid, in conjunction with Proteinase, bone marrow serine (flexizyme), is incorporated into the 3' end of the orthogonal tRNA of purifying by use.Or, also can use by chemical reaction or enzyme reaction, special acid is incorporated into the tRNA of tRNA.
peptide storehouse
In this manual, peptide storehouse refers to and comprises peptide of more than two kinds, to be imported with above-mentioned crosslinking structure, in other words comprises the library of the different peptide of more than two kinds of sequence.Form in each peptide in peptide storehouse, the amino acid except the amino acid having sulfydryl except side chain and the special acid represented by formula (I) can be arbitrary amino acid.Therefore, such as, for the peptide be made up of 25 amino-acid residues, only comprise one group of amino acid with sulfydryl and represented by formula (I) special acid time, even if only use 20 kinds of natural amino acids, also can have 20 in theory 23kind, for library, form enough sizes.
Peptide storehouse of the present invention, owing to stablizing secondary structure by being cross-linked, therefore permeability of cell membrane is also superior, utilizes this peptide storehouse to screen, then can obtain to target have high-affinity peptide or suppress the peptide etc. of function of target protein have physiologically active peptide, etc. useful peptide.
Further, as a kind of mode of peptide storehouse, each peptide can be connected with its mRNA of coding.Accordingly, become the peptide storehouse that phenotype (aminoacid sequence of peptide) is illustrated as genotype (nucleotide sequence), invitro can be applied and show.In other words, shown the displaying storehouse of (displaying) by the peptide of its translation product from genetic information, select peptide.Accordingly, the label each random peptide molecule in library is added with by biological method amplification and reading is caused.
Invitro shows, refers to that the peptide utilizing cell free translation system (being also called invitro translation system) and synthesis is mapped with genetic information and the method for showing, knownly comprises ribosomal display, mRNA shows, DNA displaying etc.Further, RAPID also can be utilized to show (unexposed).No matter any displaying method, all has following mechanism: by the genetic information be recorded on mRNA or DNA being connected with the peptide of encoding according to its genetic information, thus as [genetic information]-[translation product] mixture and be mapped.In ribosomal display, mRNA-rrna-peptide, this three forms mixture.In mRNA shows and RAPID shows, form the mixture of mRNA-peptide.In DNA shows, form the mixture of DNA-peptide.In the present invention, arbitrary invitro can be utilized to show storehouse.
the manufacture method in peptide storehouse
Although for the manufacture method in library of peptide with stochastic sequence, be not particularly limited, but such as can in cell free translation system, the template nucleic acid (DNA of mRNA or correspondence) having a stochastic sequence according to the region at encoded peptide carries out translation synthesis and obtains.
Therefore, a kind of mode of the manufacture method in peptide storehouse, comprises following operation:
I () manufactures mRNA library, this mRNA library has and determines that side chain has the amino acid whose codon of sulfydryl and determines the group of the codon of special acid represented by formula (I) in the RNA of coding random amino acid sequence, in each group, interval 2,3,6 or ten amino acid unit and configuring determines that side chain has the amino acid whose codon of sulfydryl and determines the position of codon of the special acid represented by formula (I);
(ii) utilize the cell free translation system translation mRNA comprising the tRNA being connected to described special acid, obtain the aggregate of the peptide being configured with described special acid in stochastic sequence; And
(iii) in each peptide, the side chain bonding of the special acid of sulfydryl and formula (I) is formed crosslinking structure.
In order to build the crosslinked peptide storehouse with stochastic sequence, in the region of the encoded peptide in mRNA sequence, configuring and repeating by multiple triplet the amino acid whose codon that the stochastic sequence that forms and decision become cross-linking precursor.Such as, as shown in Figure 5, the special acid to side chain with propargyl chloride structure distributes an extension codon, the position only separating the codon base of the stochastic sequence of suitable amino acid unit (as mentioned above, 2,3,6 or ten amino acid unit) therefrom such as, import halfcystine.
Further, the template mRNA being configured with the codon base of the stochastic sequence of random length except being assigned with these two amino acid whose each codons and initiator codon is built.By translating this mRNA, by be positioned at the sulfydryl of cysteine side chain, with the nucleophilic substitution reaction of propargyl chloride and build thioether bond, the crosslinked peptide storehouse with stochastic sequence can be built.
In order to build the library with stochastic sequence, usually, in order to make 20 kinds of all Argine Monohydrochlorides occur at random, by NNK (N represents any base selected from G, A, C or U, and K represents U or G) as the Codon sequences of mRNA becoming its template.But, when the special acid of the formula (I) of the electrophilic reagent that will become for the formation of crosslinking structure is suitable for an extension codon, such as time AUG (AUG as initiator codon and can extend codon these two kinds uses), with NNK library construction peptide storehouse, then owing to can occur at random as the one of the codon represented by NNK, AUG, therefore multiple above-mentioned special acid can be included in peptide storehouse in.
Therefore, during using AUG as the codon of special acid of the formula of decision (I), repeat when using the triplet be made up of NNC or NNU (N represents any one base in A, U, G, C), during as stochastic sequence, AUG can be made to there will not be in stochastic sequence, the special acid of introducing-type (I) on the position that can only specify in stochastic sequence.
When using the library of the base of NNU or NNC stochastic sequence, there will not be 5 seed amino acids (Met, Trp, Gln, Lys, Glu).When favourable part when making these amino acid occur exceedes disadvantage when this special acid is configured in beyond the position of expectation, on NNU, NNC basis, also can use NNK further.
Further, by the codon corresponding with the amino acid that there will not be when using NNU or NNC, can also be used in and import to the position of specifying by for the formation of other special acids of crosslinking structure or the special acid with other additional functions.Such as, on AUG basis, these four kinds of codons of UGG, CAG, AAG, GAG can also be utilized further, above-mentioned special acid is imported to the position of specifying.
For the mRNA containing NNU, NNC, NNK, the DNA that such as can be synthesized containing NNT, NNC, NNK by various DNA synthesizer, is transcribed this DNA and obtains.
In above-mentioned exemplary a kind of mode, the special acid of formula (I) is imported in peptide chain extension reaction by extension tRNA.At this, initial tRNA matches with the AUG codon of translation starting position and the amino acid be connected is imported to the N-end of peptide, but the AUG codon of position in addition matches with the extension tRNA with CAU codon.Therefore, AUG codon is corresponding with two seed amino acids by two kinds of tRNA.In this specification sheets, the AUG codon matched with initial tRNA is called initial AUG, and with extend the AUG codon that matches with tRNA simply referred to as AUG.
In the present invention, in the cell free translation system be made up of composition most preferred according to object, utilize the DNA corresponding with the base sequence becoming translation template or RNA molecule further.In nucleotide sequence, identically with utilizing the protein expression system of viable cell, on the basis in the region of coding object aminoacid sequence, can add according to the translation system used and comprising the base sequence favourable to translation.Such as, when use utilizes the ribosomal system of Escherichia coli, by making the upstream of initiator codon comprise Shine-Dalgarno (SD) sequence or Epsilon sequence etc., the efficiency of translation reaction is improved.
On the N-terminal in the region of encoded peptide, configuration initiator codon.Initiator codon is triplet sequences AUG normally.But, in the initial tRNA synthesized by invitro responsive transcription, by using arbitrary sequence as anticodon sequence, can reprogrammed initiator codon, thus on AUG codon basis, other base sequence can also be utilized as initiator codon.
In order to invitro shows, C-terminal side comprises the sequence for being connected with as the peptide of its translation product by nucleic acid molecule.Such as, when the mRNA that utilization employs tetracycline connexon (puromycinlinker) shows method, can the mRNA being connected to tetracycline connexon in mRNA storehouse in advance be joined in translation system, form the mixture library of mRNA-peptide thus.In order to make tetracycline effectively be attached on ribosomal A site, between the 3' end side of usual mRNA and tetracycline, be inserted with connexon.
The substrate (substrate) (aminoacyl tRNA analogue) of tetracycline as transpeptidation reaction on rrna works, and by being incorporated into the C-terminal of extension peptide, is connected by mRNA with peptide.MRNA shows that method to be a kind ofly connected via suitable connexon with peptide by mRNA by invitro translation system, thus by genotype and phenotype all-in-one-piece technology, as long as this object can be reached, also can replace tetracycline and utilize the connexon with the material of said function comprising other, this belongs to the scope that those skilled in the art know.
Further, as additive method, the mRNA not using and be connected to connexon in advance can also be utilized, and the hybridization of the connexon passed through in invitro translation system and mRNA, form the method in the mixture library of mRNA-peptide.Such as, by will the phenylalanine connexon (Phenylalaninelinker) (3'-phenylalanine-ACCA-PEG-[with the base sequence of the 3' terminal regions complementary in mRNA storehouse]-5') prepared in conjunction with Proteinase, bone marrow serine be utilized to form complementary strand with mRNA storehouse, form the mixture library (being recorded in " RAPID shows method " of undocumented application PCT/JP2010/68549) of mRNA-peptide.Now, the encode downstream (3' stub area) in region of peptide of mRNA comprises base sequence for hybridizing with connexon.
In specific embodiment described later, at the N-terminal configuration initiator AUG codon of peptide, the extension AUG codon (or codon UGC of coding Cys) of the special acid of configuration codes formula (I) after separating the stochastic sequence being equivalent to several amino-acid residue in way, after continuing the stochastic sequence of the suitable amino acid unit of configuration further, the codon UGC (or aforementioned codon of the special acid of coding type (I)) of configuration codes Cys, continues to be configured to C-terminal by stochastic sequence further.Please refer to Fig. 2 and Fig. 5.And then the codon that configuration codes becomes the GlySerGlySerGlySer of connexon is continued afterwards.
based on the aminoacylation in conjunction with Proteinase, bone marrow serine
The RNA enzyme (ARS ribozyme) of amino acid substrate acidylate to the function of arbitrary tRNA of the structure had having expectation in conjunction with Proteinase, bone marrow serine (flexizyme).Different from natural ARS proteolytic enzyme in conjunction with Proteinase, bone marrow serine, not there is specificity to each amino acid and each tRNA, can realize using the aminoacylates of the arbitrary amino acid beyond the amino acid that originally should connect.Specifically, because amino acid whose recognition site does not have the substituting group of α position, be therefore not limited to L amino acid, and the acid of alcohol acid (α position is hydroxyl), α-N-methylamino-, α-N-acylamino acid, D-amino acid etc., also can be used as substrate.Further, as adorned amino acid after the translation such as ε-N-acetyllysine or ε-N-methyllysine, also can as substrate.Detailed content except aforesaid about in conjunction with except the document of Proteinase, bone marrow serine, also be recorded in Y.Goto, H.Suga (2009) " TranslationinitiationwithinitiatortRNAchargedwithexoticp eptides " JournaloftheAmericanChemicalSociety, Vol.131, No.14,5040-5041; WO2008/059823 " N-terminal has translation synthesis and the application thereof of the polypeptide of non-natural backbone "; Goto etc., ACSChem.Biol., 2008,3,120-129; T.J.Kang, Deng, Chem.Biol., 2008,15,1166-1174 " ExpressionofhistoneH3tailswithcombinatoriallysinemodific ationsunderthereprogrammedgeneticcodefortheinvestigation onepigeneticmarkers "; WO2008/117833 " synthetic method of cyclic peptide compound " etc.
In the present invention, use is added in cell free translation system in conjunction with Proteinase, bone marrow serine with orthogonal (Orthogonal) tRNA of special acid acidylate, import special acid to peptide sequence thus.
Orthogonal tRNA; refer to that ARS owing to can not be translated endogenic natural origin in system (such as; derive from colibacillary ARS proteolytic enzyme) identify; therefore can not by aminoacylates in translation system, but effectively can express in the peptide symthesis reaction on rrna and to match with the codon of mRNA and by the amino acid whose tRNA determined.As orthogonal tRNA, such as, can use and derive from natural suppression tRNA (suppressortRNA) not of the same race or artificial constructed tRNA.As mentioned above, in the present invention, in order to import special acid, the orthogonal tRNA of manual transcription product preferably can be used.
Having activated amino acid ester in conjunction with Proteinase, bone marrow serine is substrate; identify carbonyl and the amino acid side chain of amino acid whose reaction site or depart from the aromatic nucleus of base and be present in the 5'-RCC-3' Sequence (R is A or G) of tRNA3' end, acidylate is to the enzyme function of the adenosine of 3' end.Not there is specificity in conjunction with the anticodon part of Proteinase, bone marrow serine to tRNA.That is, no matter change the anticodon part of tRNA into any sequence, also can not affect aminoacylates efficiency.By in conjunction with Proteinase, bone marrow serine, arbitrary special acid can be connected to there is arbitrary anticodon sequence tRNA on, therefore, it is possible to arbitrary special acid is mapped with arbitrary codon.Thus, the library being imported with arbitrary special acid can be prepared.
Below, the known structure in conjunction with Proteinase, bone marrow serine (RNA sequence) is shown.
Prototype is in conjunction with Proteinase, bone marrow serine (originalflexizyme) Fx
[GGAUCGAAAGAUUUCCGCAGGCCCGAAAGGGUAUUGGCGUUAGGU-3',45nt](SEQIDNO:1)
Dinitrobenzene benzyl is in conjunction with Proteinase, bone marrow serine (dinitrobenzylflexizyme) dFx
[5'-GGAUCGAAAGAUUUCCGCAUCCCCGAAAGGGUACAUGGCGUUAGGU-3',46nt](SEQIDNO:2)
Enhancement type is in conjunction with Proteinase, bone marrow serine (enhancedflexizyme) eFx
[5'-GGAUCGAAAGAUUUCCGCGGCCCCGAAAGGGGAUUAGCGUUAGGU-3',45nt](SEQIDNO:3)
Amino is in conjunction with Proteinase, bone marrow serine (aminoflexizyme) aFx
[5'-GGAUCGAAAGAUUUCCGCACCCCCGAAAGGGGUAAGUGGCGUUAGGU-3',47nt](SEQIDNO:4)
Different from natural ARS proteolytic enzyme in conjunction with Proteinase, bone marrow serine; skip as aminoacylation first step, generate high energy intermediate (aminoacyl AMP) process; the only cohesive process to tRNA of catalytic amino acid substrate; therefore as amino acid substrate, need to use in advance by the amino acid of weak activation.That is, skip amino acid whose adenylylation, therefore replace this process and be used in the amino acid derivative of the ester bond in the carbonyl carrying out acidylate with weak activation.Usually, the activation of acyl group is realized by the ester bond with electron-withdrawing group, but has the ester of strong electron-withdrawing group, is not only hydrolyzed in water, also causes the acidylate to random rna simultaneously.Therefore, as amino acid substrate, need the amino acid using weak activation, be difficult to occur with these side reactions under the state not having enzyme.This weak activation, such as, can use AMP, cyanomethyl ester (cyanomethylester), thioesters or the benzyl ester with other electrophilic functional groups such as nitro or fluorine etc. and carrying out.
Further, amino acid substrate needs to have aromatic nucleus, with combined Proteinase, bone marrow serine identification in amino acid side chain or disengaging base.Activated amino acid ester, be have as the aminoacyl AMP in natural aminoacylation by the ester bond of weak activation, and at amino acid side chain or depart from base the amino acid substrate with aromatic nucleus.As the example of suitable activated amino acid ester, can enumerate, aminoacyl-cyanomethyl ester (CME:cyanomethylester), aminoacyl-dinitrobenzene benzyl ester (DNB:3,5-dinitrobenzylester) or aminoacyl-4-chlorobenzyl thioesters (CBT:p-chloro-benzylthioester) etc., but be not limited to these.
Such as, in embodiment described later, N α-ethanoyl-L-Phe (Ac-F) is connected to artificial constructed initial tRNA, carry out the importing to peptide N-terminal, simultaneously, side chain had the own-4-acetylenic acid ((s)-2-amino-6-chlorohexy-4-ynoicacid) of (s)-2-amino-6-chlorine of propargyl chloride, (s)-2-amino-7-chlorine-5-in heptan acetylenic acid ((s)-2-amino-7-chlorohepto-5-ynoicacid), s three seed amino acids (please refer to the structural formula under the table 1 in embodiment) of ()-2-amino-8-chlorine pungent-6-acetylenic acid ((s)-2-amino-8-chloroocto-6-ynoicacid) are connected respectively to artificial constructed extension tRNA.For these three kinds of compounds, respectively own for (s)-2-amino-6-chlorine-4-acetylenic acid DNB, (s)-2-amino-7-chlorine-5-in heptan acetylenic acid DNB, (s)-2-amino-8-chlorine pungent-6-acetylenic acid DNB are used as substrate, mixing dFx and tRNA, can prepare the tRNA be combined with these compounds.On the other hand, for N α-ethanoyl-L-Phe, by being used as substrate by N α-ethanoyl-L-Phe CME, mixing eFx and tRNA, can prepare N α-ethanoyl-L-Phe in conjunction with tRNA.
[table 3]
Based on the acylation reaction in conjunction with Proteinase, bone marrow serine, can carry out in the solution, also can utilize and use the post (column) of the ARS ribozyme be fixed on carrier and react.Such as; if a small amount of at 100 below μ l of reaction scale (scale) of translation; then carry out the acidylate based on the tRNA in conjunction with Proteinase, bone marrow serine in the solution; the throw out (pellet) reaction soln being carried out to alcohol settling is dissolved in suitable damping fluid (such as; the Potassium ethanoate, pH5 etc. of 1mM) in, add reactive system to.For reaction conditions, as long as select suitable condition, but as reaction conditions during a small amount of scale one example, can by comprise final concentration be 0.5 ~ 20 μM tRNA, 0.5 ~ 20 μM in conjunction with the amino acid substrate of Proteinase, bone marrow serine, 2 ~ 10mM, the MgCl of 0.6M 2, the 0.1M reaction buffer of pH7.5, under 0 degree (DEG C), react 1 ~ 24 hour.
If translation reaction scale more than 100 μ l, then consider the recycling in conjunction with Proteinase, bone marrow serine, more preferably use be fixed on carrier in conjunction with Proteinase, bone marrow serine.As carrier, such as, also can use resin, agarose, sepharose, magnetic bead etc., but be not particularly limited.Will in conjunction with Proteinase, bone marrow serine be fixed on carrier reacts time, such as, can historically in Murakami, H., Bonzagni, and Suga, H. (2002) N.J.. the method for " Aminoacyl-tRNAsynthesisbyaresin-immobilizedribozyme. " J.Am.Chem.Soc.124 (24): 6834-6835 is carried out.As the separation of the aminoacylates tRNA of reaction product, can be undertaken by various method.As a kind of example, comprise the method for damping fluid from post wash-out that use contains the EDTA of about 10mM.Securing the resin of ARS ribozyme, such as, by balancing with reaction buffer, then can reclaim use more than ten time.
initial tRNA and extension tRNA
In natural translation reaction, the beginning of initial tRNA only for translating, and be not used to extension, on the contrary, extend and be not used to initial action with tRNA, this is very important.The difference of this initial tRNA and extension tRNA, also identical in the present invention.
In this application, in order to acidylate special acid, preferably use artificial tRNA.As a kind of indefiniteness example of the artificial tRNA of extension tRNA, comprise tRNA asn-E2.The base sequence of this tRNA is based on as the natural tRNA deriving from colibacillary extension tRNA asn(5'-UCCUCUG s4uAGUUCAGDCGGDAGAACGGCGGACUQUU t6aAYCCGUAU m7gUCACUGGTYCGAGUCCAGUCAGAGGAGCCA-3') (SEQIDNO:7) ( s4u:4-thio uridine (4-thiouridine), D: dihydrouridine (dihydrouridine), Q: pigtail glycosides (queuosine), t6a:6-threonyl formamyl VITAMIN B4 (6-threonylcarbamoyladenine), Y: bosom Russia's glycosides (wyosine), m7g:7-methylguanosine (7-methylguanosine), T: thymine ribonucleoside (ribothymidine)).Inventors of the present invention, to this natural tRNA, remove modified base, and import sudden change, are transcribed thus by invitro, and preparation is not by the tRNA as extension tRNA of colibacillary 20 kinds of L-Aminoacylase aminoacylations asn-E2.NNN place is equivalent to anticodon, changes corresponding codon into.
(tRNA asn-E2: 5'-GGCUCUGUAGUUCAGUCGGUAGAACGGCGGACU nN naAUCCGUAUGUCACUGGUUCGAGUCCAGUCAGAGCCGCCA-3'(SEQIDNO:5), [part totally 8 places modified are removed. s4U8U、D16U、D20U、 t6A37A、Y39U、 m7G46G、T54U、Y55U。About the 34th Q, be anticodon, therefore corresponding codon and change] [Mutational part totally 4 places.U1G、C2G、G71C、G72C])
As a kind of indefiniteness example of the artificial tRNA of initial tRNA, comprise tRNA fMet.The base sequence of this tRNA is based on colibacillary natural tRNA fMet(5'-CGCGGGG s4uGGAGCAGCCUGGDAGCUCGUCGGGCmU cAU(Cm:2'-O-methylcytidine (2'-O-methylcytidine)) of AACCCGAAGAUCGUCGGTYCAAAUCCGGCCCCCGCAACCA-3') (SEQIDNO:8).Inventors of the present invention, to this natural tRNA, are removed modified base, are transcribed, prepare the tRNA first of 5' end C being changed into the initial action tRNA of G by invitro fMet.CAU place is equivalent to anticodon, corresponding initiator AUG codon.(the tRNA used in the application fMet: 5'-GGCGGGGUGGAGCAGCCUGGUAGCUCGUCGGGCU cAUaACCCGAAGAUCGUCGGUUCAAAUCCGGCCCCCGCAACCA-3'(SEQIDNO:6), [part totally 6 places modified are removed. s4U8U、D20U、Cm32C、T54U、Y55U。] [Mutational part totally 1 place.C1G]) part and parcel is first base (natural tRNA of 5' end in initial tRNA fMetin be the tRNA of C, the application fMetin be G) with the 72nd base (natural tRNA fMetand the tRNA of the application fMetin be A), do not form complementary strand.According to this incomplementarity chain, sometimes formyl radical is made to transfer to Met-tRNA by methionyl transformylase (methionylformyltransferase) (MTF) fMetbut (, nonsensical when this part utilizes initial special acid), and sometimes suppressed with the combination of EF-Tu.
In embodiment described later, N α-ethanoyl-L-Phe is connected to tRNA fMet cAU, import to peptide N-terminal, meanwhile, three kinds of special acids side chain with propargyl chloride are connected respectively to tRNA asnE2 cAU.TRNA asn-E2 nNNanticodon sequence (NNN, N represent arbitrary base) can be made into various sequence and uses, but when determining that the change codon that side chain has a special acid of propargyl chloride structure is AUG, anticodon sequence becomes CAU.
Because these initial use and the artificial tRNA that extends have orthogonality to natural A RS, therefore can not be connected with natural amino acid in translation system, but when translation initiation reaction, peptide chain extension reaction on rrna, can accept without any problems.These artificial tRNA are confirmed, can actually be used in the concrete cell free translation system used in embodiment.But operable artificial tRNA is not limited thereto in the present invention.As long as those skilled in the art, should understand in the present invention and can be suitable for according to the composition of used cell free translation system selecting to import special acid and available tRNA.
invitro screens
In the present invention, expressing polypeptide storehouse constructed in cell free translation system can be applicable to as display technique such as invitro such as mRNA displayings grade completely, therefore can from by up to 10 13the peptide molecule be combined with target is formulated in the expressing polypeptide storehouse of planting above highly diverse formation.
Invitro display technique as Engineering of Molecular Evolution instrument and be utilized.In Engineering of Molecular Evolution, have for the purpose of the function of expectation or the protein of character or peptide to formulate, prepare the potential gene of tool on a large scale, therefrom select the phenotypic clone with expectation.Carry out roughly as follows: initial preparation DNA colony (DNA library), obtains RNA colony (RNA library) as invitro transcription product, obtain peptide colony (peptide storehouse) as invitro translation product.Select that there is the function of expectation or the peptide of character from this peptide storehouse by certain screening system.
Such as, if wish to obtain the peptide molecule with specific protein bound, Shi Tai colony flows into the post securing target proteins, can reclaim the mixture of the peptide molecule be combined with pillar.Now, by invitro display technique, on each peptide molecule, similar label is added with its template nucleic acid molecule like that.If mRNA display libraries, each peptide molecule is added with mRNA.Therefore, turn back to DNA from the colony of the peptide-mRNA mixture reclaimed by ThermoScript II, by pcr amplification, obtain the subjective library comprising the phenotypic clone with expectation in a large number of thinking, and then carry out same choice experiment.
Or, in order to avoid reclaiming the possibility of RNA fit (RNAaptamer), can also become for the purpose of double-strand (DNA RNA hybrid (DNA/RNAhybrid)) by nucleic acid moiety, carrying out reverse transcription reaction before the selection.By repeating this operation, the phenotypic clone along with passing from generation to generation with expectation is concentrated in colony gradually.
When qualification peptide is fit, invitro display libraries is mixed with target substance, select molecule (spike) that display is attached to the peptide of target substance, that be mapped, repeat the operation being prepared nucleic acid library from the nucleic acid moiety of the selected molecule be mapped by PCR, the gene that the peptide that is combined with target substance is fit can be cloned.
As target substance, being generally any mixture of albumen, peptide, nucleic acid, carbohydrate, lipid, these materials, also can be other any compounds.
In order to select spike, [genetic information]-[peptide] mixture is needed to contact with target substance, and by suitable method, other the most mixtures be never combined with target substance, Separation and Recovery shows the mixture of the peptide be combined with target substance.As this recovery method, existing multiple known method.
Such as, if implemented in advance by combining and callable modification to solid phase target substance, then can become convenient.Such as, on target substance, connect polyhistidine label in advance, the specific binding to the carrier supported by Ni-NTA of polyhistidine label can be utilized and reclaim.As this specific binding, except the combination of polyhistidine peptide/metal ion (nickel, cobalt etc.), also can utilize biotin-binding protein (avidin, streptavidin etc.) combination such as/vitamin H, maltose binding protein/maltose, glutathione-S-transferase/gsh, antibody/antigen (epi-position), but be not limited to these.
The present invention includes the invitro screening by being repeated below, formulate the expressing polypeptide combined with target substance: contacted with target substance in peptide storehouse, select the spike showing the peptide be combined with target substance, the nucleotide sequence of the spike selected by amplification, selects spike from the library of the peptide again synthesized cell free translation system for template with the nucleotide sequence of amplification.Especially, owing to using by integrating crosslinking structure to linear peptides, to make the library of the more peptide that exist for the library of object, that comprise secondary structure guidance quality of specific secondary structure being called spirane structure, therefore there is the tertiary structure thinking suitable to molecular interaction, thus just do not obtain and molecule that target proteins is easily combined, and can obtain and be combined with the position participating in its molecular interaction and show the peptide of inhibit activities.
The initiative of the expressing polypeptide compound be combined with target substance comprises: reclaim the spike shown with target substance binding peptide, analysis of nucleic acids sequence, peptide sequence is determined from nucleotide sequence, select suitable expressing polypeptide based on obtained peptide sequence, obtain aminoacid sequence and the nucleotide sequence of the expressing polypeptide be combined with target substance thus.Further, based on the sequence information obtained, arbitrary method can be used, synthesis expressing polypeptide, and carry out purifying and separation.Use the peptide obtained, carry out the combining assessment of target proteins or the confirmation of inhibit activities, highly active expressing polypeptide can be obtained.
Therefore, the present invention includes the method obtaining the peptide molecular interaction of target proteins to inhibit activities from peptide storehouse, comprise the following steps:
(first) selects the primary screening of the peptide be combined with target proteins;
(second) evaluates the binding ability by the peptide selected by primary screening, determines that described peptide is the postsearch screening of the peptide showing reliable binding ability,
Described primary screening step comprises operation:
I () prepares the library comprising the peptide with secondary structure guidance quality;
(ii) peptide storehouse is contacted with target proteins; And
(iii) peptide molecule be combined with target proteins is selected.
build the method containing spiral library when Bcl-2 is target and obtain phase between Inhibitory molecules from it the method of the peptide of mutual effect
Below, to when the construction process of the Bcl-2 enumerated in the item of embodiment described later as peptide storehouse during target proteins and the inhibiting peptide preparation method from it are described (Fig. 4).Mode illustrated is here only exemplary, the invention is not restricted to which.
target proteins Bcl-2
Apoptosis is the programmed death of cell, has various path, as its a kind of regulatory mechanism having Bcl-2 albumen and participate in.Bcl-2 albumen suppresses it active by the protein binding with cell death inducing.Known because Bcl-2 albumen is more at cancerization cells, therefore apoptosis is difficult to be induced.
Therefore, as long as obtain with Bcl-2 highly selective and the powerful molecule combined, then this molecules in inhibiting Bcl-2 is combined with Cell Apoptosis Inducing Protein, can to cancerization cell transfered cell apoptosis.Therefore, although study this Inhibitory molecules as possible in recent years, the research and development of the interactional molecule between this arrestin are comparatively difficult.Due to the spirane structure being called BH3 region that this molecular interaction utilizes Bcl-2 albumen to comprise, as long as be therefore considered to form the library with the molecule of spirane structure, then can obtain highly selective from this library and the inhibitor of brute force, thus also carry out the research of the chemical libraries from the aforesaid crosslinked peptide based on alkene formation.But, fail to obtain the molecule with powerful inhibit activities.
the structure in mRNA storehouse
First, in order to build peptide storehouse by translation, preparation becomes the mRNA storehouse of template.The length of the sequence of the part be translated of mRNA is arbitrary.Such as, can be the sequence (such as, Fig. 5 from pond 16-1 to 24-4) of the length comprising 16 ~ 24 codons.Wherein, N-terminal is initiator AUG codon.Further, the codon that coding becomes the GlySerGlySerGlySer of connexon can also be added in C-terminal side.Initiator AUG codon downstream can be that (N represents any one base in A, U, G, C respectively for the random codon sequence of NNU or NNC.)。Specific 1 codon in this stochastic sequence is designed to the special acid that AUG is used for introducing-type (I), from this codon separate 2,3,6 or ten amino acid unit position the codon UGC (such as, Fig. 2 and Fig. 5) of configuration codes Cys.
the structure in peptide storehouse
The mRNA storehouse of acquisition is translated based on altered genetic codon table.Such as, build the translation system removing methionine(Met) from common 20 seed amino acids, replace the methionine(Met) removed, utilize (i) at tRNA fMet cAUupper connection N αthe aminoacylates tRNA of-acetylated amino acids, (ii) are at tRNA asnE2 cAUupper connection side chain has the aminoacylates tRNA of the special acid of propargyl chloride structure, translation template mRNA.These 2 aminoacylates tRNA can utilize and prepare in conjunction with Proteinase, bone marrow serine.
At this, the aminoacylates tRNA of (i) is identified as initiation factor and corresponding initiator AUG codon, and the aminoacylates tRNA of (ii) is identified as elongation factor and corresponding A UG codon.Therefore, only remove methionine(Met), just can import two seed amino acids to AUG codon.In the peptide be translated, act on propargyl chloride structure by the sulfydryl of halfcystine and the thioether bond that obtains, form crosslinking structure.
Further, by tetracycline being incorporated into the 3' end of each mRNA in mRNA storehouse, thus after translation, the C-terminal of peptide is connected with mRNA via Pu (tetracycline).During beginning, also can utilize except N αarbitrary special acid outside-phenyl methyl ketone-L-Phe or methionine(Met) or other 19 kinds of Argine Monohydrochlorides.
the acquisition of inhibiting peptide
By mRNA, obtained peptide storehouse is shown that the various invitro such as method or ribosomal display method shows that method is screened, select the peptide be combined with Bcl-2.
Due to use to be designed in translated polypeptide stable formed with the interactional spirane structure as BH3 region of target proteins, the peptide storehouse of secondary structure guidance quality, even if therefore only screen to be combined into index, the peptide obtained is combined with the molecular recognition site of target proteins, suppresses the possibility of its molecular interaction also higher.
to the structure in the peptide storehouse of different target
The invention is not restricted to BCl-2, and can be applicable to various molecule be the screening of target.As target molecules, be preferably molecular interaction recognition helix structure and the molecule that carries out.The molecule of molecular interaction is carried out as recognition helix structure, such as can enumerate p53 or hDM2 (F.Bernal, A.F.Tyler, S.J.Korsmeyer, L.D.Walensky, G.L.VerdineJ.Am.Chem.Soc.1292456-2457 (2007) .), but be not limited thereto.
Below, the present invention is illustrated by embodiment.Wherein, these embodiments are for illustration of of the present invention, instead of are used for limiting scope of the present invention.
Embodiment
Be combined with the molecular interaction position in the BH3 region based on Bcl-2 to obtain and suppress the peptide of its molecular interaction, build the peptide storehouse (NNUmRNA storehouse) in sequence with the crosslinking structure formed by special acid and halfcystine, show that method is selected by mRNA.
nNUmRNA storehouse
First, preparation has the double-stranded DNA (following, only to record forward (Forward) chain with 5' → 3' order) of following sequence.
TAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACAT(ATG)(NNT)s(ATG)(NNT) 3(TGC)(NNT)t(GGT)(AGC)(GGC)(AGC)(GGC)(AGC)(TAG)GACGGGGGGCGGAAA
Translated region codon in () expansion.N represents any one in A, T, G, C.S and t represents the repeat number of triplet, is equivalent to 2 and 8 or 6 and 4 or 2 and 12 or 6 and 8 or 10 and 4 or 2 and 16 or 6 and 12 or 10 and 8 or 14 and 4 respectively.
Then, utilize this double-stranded DNA of T7 polymerase transcription, obtain the library that the mRNA pond (mRNApools) that represented by following sequence forms and (please refer to the table of the bottom of Fig. 5.)。
GGGUUAACUUUAAGAAGGAGAUAUACAU(AUG)(NNU)s(AUG)(NNU) 3(UGC)(NNU)t(GGU)(AGC)(GGC)(AGC)(GGC)(AGC)(UAG)GACGGGGGGCGGAAA
The above-mentioned transcription product of balanced mix, is used in following experiment.
mRNA shows
By repeatedly carrying out the circulation from following " with the connection of tetracycline connexon " to " amplification of the sequence information of the peptide of recovery ", select the peptide (Fig. 4) be combined with Bcl-2 from random peptide library.
with the connection of tetracycline connexon
Annealing in the tetracycline connexon represented by following sequence and above-mentioned mRNA storehouse, is connected by T4RNA ligase enzyme (T4RNALigase).SPC18 represent carbon atom and Sauerstoffatom add up to 18 PEG.
pdCTCCCGCCCCCCGTCC(SPC18) 5CC(Pu)
translation
The mRNA (Fig. 6) be connected with connexon is translated based on altered genetic codon table.In the present embodiment, build the translation system removing methionine(Met) from common 20 seed amino acids, replace the methionine(Met) removed, add use prepare in conjunction with Proteinase, bone marrow serine, two kinds of aminoacyl tRNA of following (i) and (ii) and translating:
I () is at tRNA fMet cAUupper connection N αthe aminoacyl tRNA of-ethanoyl-L-Phe,
(ii) at tRNA asnE2 cAUabove to connect respectively
Own-4-acetylenic acid ((s)-the 2-amino-6-chlorohexy-4-ynoicacid) (X of (s)-2-amino-6-chlorine 0),
(s)-2-amino-7-chlorine-5-in heptan acetylenic acid ((s)-2-amino-7-chlorohepto-5-ynoicacid) (X 1),
Pungent-6-the acetylenic acid ((s)-2-amino-8-chloroocto-6-ynoicacid) of (s)-2-amino-8-chlorine (X 2) aminoacyl tRNA.
Then translate, form thioether bond, synthesizing cross-linked peptide storehouse between the propargyl chloride structure comprised at these special acids and the sulfydryl of halfcystine, the C-terminal constructed at peptide is combined with the mixture of mRNA via Pu.
the acquisition of the peptide be combined with Bcl-2
To the crosslinked peptide storehouse that the Bcl-2 being fixed on TALON pearl (TALONbeads) is mixed with, stir 30 minutes at 4 DEG C.Magnet is utilized to remove supernatant, with the magnetic particle that buffer solution for cleaning is remaining.Add PCR solution to pearl, 95 DEG C are heated 5 minutes, slough peptide from pearl, reclaim supernatant.
the amplification of the sequence information of the peptide reclaimed
Peptide-the mRNA reclaimed being combined with Bcl-2 is DNA amplification by reverse transcription PCR.Transcribe acquisition DNA and as mRNA.
the qualification of the peptide sequence selected
A repeatedly above-mentioned series of operations, when the rate of recovery of peptide-mRNA reaches capacity, uses the DNA of amplification to carry out TA clone, identifies the peptide sequence obtained.
result
If translation NNUmRNA storehouse, AUG codon translation then in stochastic sequence becomes side chain to have the special acid of propargyl chloride, form covalent linkage between the sulfydryl being encoded in the halfcystine of the position from this separation for amino acids 4 amino acid unit, generate crosslinked peptide storehouse.Use this peptide storehouse and carry out mRNA displaying, its result, the 3 kinds of special acids no matter using the length of the connexon between propargyl chloride from amino acid backbone structures different, that is, the own-4-acetylenic acid (X of (s)-2-amino-6-chlorine 0), (s)-2-amino-7-chlorine-5-in heptan acetylenic acid (X 1), the pungent-6-acetylenic acid of (s)-2-amino-8-chlorine (X 2) 3 kinds of special acids in peptide storehouse arbitrarily in, the 7th time time, the rate of recovery of mRNA all reaches saturated.
Therefore, identify the peptide sequence after 6 times, its result is learnt, most sequence has crosslinking structure, has the peptide (table 1) of 2 l-asparagines near the central authorities obtaining its sequence.In concentrated peptide sequence, can find the apokoinou construction with two l-asparagines, this shows to show method by mRNA, obtains the interactional molecule suppressed with Bcl-2 as scheduled.
[table 4]
Table 1
From above result, can confirm to utilize the peptide containing peptide crosslinking structure, invitro shows that method plays function effectively.
the evaluation of the Bcl-2 binding ability of the peptide selected
Then, be there is by Fmoc Solid phase synthesis the crosslinked peptide of the aminoacid sequence illustrated in above-mentioned table, calculate dissociation constant (KD) according to the evaluation assessment that make use of SPR (SurfacePlasmonResonance, surface plasma resonance), evaluate binding ability.
Specifically, first, will containing NiCl 2solution spill substrate to being combined with Ni, be then spilled into the Bcl-2 albumen that C-terminal has Histag, ankyrin on substrate thus.Then, add the solution of the peptide comprising solid phase synthesis in turn with suitable concentration, now read the variations in refractive index according to the weight on substrate, calculate this peptide in conjunction with speed and to dissociate speed, evaluate binding ability thus.The results are shown in following table.
[table 5]
Learn, the peptide of synthesis all illustrates the value less than KD=1nM, and powerful binding ability is shown.
The free content of sequence table
Sequence number 1: in conjunction with Proteinase, bone marrow serine Fx
Sequence number 2: dinitrobenzene benzyl is in conjunction with Proteinase, bone marrow serine dFx
Sequence number 3: enhancement type is in conjunction with Proteinase, bone marrow serine eFx
Sequence number 4: amino is in conjunction with Proteinase, bone marrow serine aFx
Sequence number 5:tRNA asn-E2
Sequence number 6:tRNA fMet
Sequence number 7: colibacillary tRNA asn
Sequence number 8: colibacillary tRNA fMet

Claims (22)

1. a peptide, this peptide has and obtains stable secondary structure by crosslinking structure,
Described peptide comprises the amino acid that at least one group of special acid represented in order to following formula (I) and side chain have sulfydryl,
Described peptide is formed with crosslinking structure by the thioether bond between the side chain of described special acid residue and described sulfydryl,
[chemical formula 1]
In formula, (A) represent singly-bound or from the alkylidene group, the carbonatoms that by carbonatoms are 1 ~ 10 be 2 ~ 10 alkenylene or carbonatoms be the alkynylene of 2 ~ 10; Atomicity that 1 ~ 2 carbon atom of main chain is replaced by Sauerstoffatom, nitrogen-atoms or sulphur atom, main chain is the connection base selected in the group that forms of alkylidene group, alkenylene or alkynylene of less than 10,
(B) represent from by-C ≡ C-,-C=C-,-Ar-,-Ar-Ar-,-Ar-C ≡ C-,-Ar-C=C-,-NHC (O)-,-C (O)-,-Ar-NHC (O)-, select the group of-Ar-C (O)-form, containing the group of at least one π key
(C) hydrogen atom or the alkyl selected from the group be made up of methyl, ethyl, propyl group, butyl or amyl group is represented,
X represent can by the substitution reaction based on sulfydryl the group that be substituted, from by Cl, Br, I ,-OSO 2me ,-OSO 2select, at described-OSO in the group that-Ar-R is formed 2in-Ar-R formula, R represents from by CH 3, NO 2, CF 3and the group selected in the group of H formation.
2. peptide as claimed in claim 1, in described formula (I), X is tosyl group or nitrobenzenesulfonyl.
3. peptide as claimed in claim 1 or 2, the special acid of described formula (I) represents in order to the individual arbitrarily of following formula (II) ~ (IV),
[chemical formula 2]
In formula, m represents the integer selected from 1 ~ 10, and X is from by Cl, Br, I ,-OSO 2me ,-OSO 2select, at described-OSO in the group that-Ar-R is formed 2in-Ar-R formula, R represents from by CH 3, NO 2, CF 3and the group selected in the group of H formation.
4. peptide as claimed in claim 1 or 2, the described amino acid with sulfydryl is selected from by halfcystine and the group that forms in order to the cysteine analogs that lower formula V and (VI) represent,
[chemical formula 3]
In formula, m represents the integer selected from 1 ~ 10.
5. peptide as claimed in claim 1 or 2, described special acid residue and described side chain have the amino-acid residue of sulfydryl, in each group, interval 2,3,6 or ten amino acid residue and configure.
6. a method, preparation has the peptide being obtained stable secondary structure by crosslinking structure, comprises following operation:
I () is configured with the expressing polypeptide of the amino acid that at least one group of side chain have sulfydryl and the special acid represented in order to following formula (I) in synthetic molecules by translation; And
(ii) in each described group, the side chain of the special acid of described sulfydryl and formula (I) is formed crosslinking structure by thioether bonding,
[chemical formula 4]
In formula, (A) represent singly-bound or from the alkylidene group, the carbonatoms that by carbonatoms are 1 ~ 10 be 2 ~ 10 alkenylene or carbonatoms be the alkynylene of 2 ~ 10; Atomicity that 1 ~ 2 carbon atom of main chain is replaced by Sauerstoffatom, nitrogen-atoms or sulphur atom, main chain is the connection base selected in the group that forms of alkylidene group, alkenylene or alkynylene of less than 10,
(B) represent from by-C ≡ C-,-C=C-,-Ar-,-Ar-Ar-,-Ar-C ≡ C-,-Ar-C=C-,-NHC (O)-,-C (O)-,-Ar-NHC (O)-, select the group of-Ar-C (O)-form, containing the group of at least one π key
(C) hydrogen atom or the alkyl selected from the group be made up of methyl, ethyl, propyl group, butyl or amyl group is represented,
X represent can by the substitution reaction based on sulfydryl the group that be substituted, from by Cl, Br, I ,-OSO 2me ,-OSO 2select, at described-OSO in the group that-Ar-R is formed 2in-Ar-R formula, R represents from by CH 3, NO 2, CF 3and the group selected in the group of H formation.
7. method as claimed in claim 6, described side chain has the amino acid of sulfydryl and the special acid of formula (I), in each group, interval 2,3,6 or ten amino acid residue and configure.
8. method as claimed in claims 6 or 7, the translation synthesis procedure of described (i) comprising: utilize and the special acid of formula (I) is connected to tRNA and the aminoacyl tRNA that obtains, and translation has the template mRNA of the change codon of this special acid of coding.
9. method as claimed in claim 8, the amino acid that described side chain has sulfydryl is by the special acid changing codon coding, the translation synthesis procedure of described (i) comprising: utilize this special acid special acid of formula (I) and side chain with sulfydryl to be connected to tRNA and the aminoacyl tRNA that obtains, and translation has the template mRNA of the change codon of special acid of encoding respectively.
10. method as claimed in claim 8, described aminoacyl tRNA is by utilizing the RNA enzyme with acidylate tRNA synthetic enzyme sample activity, special acid being connected to tRNA and obtaining.
11. method as claimed in claims 6 or 7, the special acid represented by described formula (I), in order to an expression arbitrarily of following formula (II) ~ (IV),
[chemical formula 5]
In formula, m represents the integer selected from 1 ~ 10, and X is from by Cl, Br, I ,-OSO 2me ,-OSO 2select, at described-OSO in the group that-Ar-R is formed 2in-Ar-R formula, R represents from by CH 3, NO 2, CF 3and the group selected in the group of H formation.
12. method as claimed in claims 6 or 7, the described amino acid with sulfydryl is selected from by halfcystine and the group that forms in order to the cysteine analogs that lower formula V and (VI) represent,
[chemical formula 6]
In formula, m represents the integer selected from 1 ~ 10.
13. 1 kinds of peptide storehouses, comprise two or more as the peptide in claim 1 to 5 as described in any one.
14. peptide storehouses as claimed in claim 13, each described peptide is connected with the mRNA of this peptide of coding.
15. 1 kinds of methods, build peptide storehouse as claimed in claim 13, comprise following operation:
I () prepares mRNA library, this mRNA library comprises the codon of special acid that at least one group of amino acid whose codon and determining determining that side chain has sulfydryl is represented by formula according to claim 1 (I) in the RNA of coding random amino acid sequence, interval 2,3,6 or ten amino acid unit in each group and be configured with and determine that side chain has the codon of the amino acid whose codon of sulfydryl and the special acid of decision formula (I);
(ii) by the cell free translation system containing the tRNA be connected with described special acid, translate described mRNA, obtain the aggregate of the peptide being configured with described special acid in stochastic sequence; And
(iii) in each peptide, make the side chain bonding of the special acid of sulfydryl and formula (I) and form crosslinking structure.
16. 1 kinds of methods, build peptide storehouse as claimed in claim 14, comprise following operation:
In operation (i) as claimed in claim 15, further, to the 3' end of mRNA in conjunction with tetracycline, obtain tetracycline in conjunction with mRNA library;
In operation as claimed in claim 15 (ii), described tetracycline is expressed in conjunction with mRNA library in cell free translation system, obtain the peptide-mRNA mixture being configured with described special acid in stochastic sequence; And
Carry out operation as claimed in claim 15 (iii).
17. methods as described in claim 15 or 16, the change codon of the special acid of decision formula (I) is AUG codon, mRNA stochastic sequence by any triplet in NNC or NNU sequence repeat form, the N in described NNC or NNU represents any one base in A, U, G, C.
18. 1 kinds of special acids represented with following formula (I),
[chemical formula 7]
In formula, (A) represent singly-bound or from the alkylidene group, the carbonatoms that by carbonatoms are 1 ~ 10 be 2 ~ 10 alkenylene or carbonatoms be the alkynylene of 2 ~ 10; Atomicity that 1 ~ 2 carbon atom of main chain is replaced by Sauerstoffatom, nitrogen-atoms or sulphur atom, main chain is the connection base selected in the group that forms of alkylidene group, alkenylene or alkynylene of less than 10,
(B) represent from by-C ≡ C-,-C=C-,-Ar-,-Ar-Ar-,-Ar-C ≡ C-,-Ar-C=C-,-NHC (O)-,-C (O)-,-Ar-NHC (O)-, the group selected the group of-Ar-C (O)-form
(C) hydrogen atom or the alkyl selected from the group be made up of methyl, ethyl, propyl group, butyl or amyl group is represented,
X represent can by the substitution reaction based on sulfydryl the group that be substituted, from by Cl, Br, I ,-OSO 2me ,-OSO 2select, at described-OSO in the group that-Ar-R is formed 2in-Ar-R formula, R represents from by CH 3, NO 2, CF 3and the group selected in the group of H formation.
19. a method, select the peptide be combined with target proteins from the peptide storehouse as described in claim 13 or 14, comprise following operation:
I peptide storehouse contacts with target proteins by (), carry out incubation; And
(ii) peptide molecule be combined with target proteins is selected.
20. a method, select the peptide molecular interaction of target proteins to inhibit activities from the peptide storehouse as described in claim 13 or 14, comprising:
(first) primary screening, comprises operation (i) as claimed in claim 19 and (ii), selects the peptide be combined with target proteins; And
(second) postsearch screening, for the inhibit activities of the molecular interaction to target proteins of the peptide selected by primary screening, is evaluated, and determines that described peptide is the peptide molecular interaction of target proteins to inhibit activities.
21. 1 kinds of methods, manufacture the peptide be combined with target proteins, comprise following operation:
I peptide storehouse as claimed in claim 14 contacts with target proteins by (), carry out incubation;
(ii) peptide-mRNA mixture be combined with target proteins is selected;
(iii) mRNA of the peptide-mRNA mixture selected by amplification, obtains peptide-mRNA mixture;
(iv) operation (i) ~ (iii) is repeated once, the peptide-mRNA mixture that concentrated affinity is high; And
(v) from the mRNA by the concentrated peptide-mRNA mixture of operation (iv), expression of peptides.
22. as the method in claim 19 to 21 as described in any one, and target proteins is the molecule of inhibited apoptosis.
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